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Hyperbranched immobilized metal affinity chromatography stationary phase and preparation method thereof

A chromatographic stationary phase and metal technology, which is applied in the field of hyperbranched immobilized metal ion affinity chromatography stationary phase and its preparation, can solve the problems of organic solvent sensitivity and inability to obtain, and achieve good hydrophilicity, improved recovery rate, and good repeatability usability effect

Inactive Publication Date: 2017-06-16
NORTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, biological macromolecules such as proteins generally exist in the form of complex mixtures, and have special properties, such as sensitivity to temperature, pH, organic solvents, etc., and separation based on their physical and chemical properties can hardly achieve good results.

Method used

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  • Hyperbranched immobilized metal affinity chromatography stationary phase and preparation method thereof
  • Hyperbranched immobilized metal affinity chromatography stationary phase and preparation method thereof
  • Hyperbranched immobilized metal affinity chromatography stationary phase and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Raw film pretreatment

[0035] Cut a whole piece of regenerated cellulose membrane into 8 small pieces, soak in methanol for half an hour to remove the grease and adhering impurities on the surface of the membrane, wash with methanol and water several times, and dry in a vacuum oven at 40°C for later use.

[0036] Preparation of Hyperbranched Immobilized Metal Ion Affinity Membrane

[0037] (1) First, introduce functional groups with amino groups on the surface of the membrane through a two-step reaction. The specific operation is: put 24 small pieces of cut original membrane into a 100mL three-necked bottle, add 40mL, 1.4M NaOH, 15 mL epichlorohydrin, React at 60°C for 2 hours. After the reaction, wash with water and methanol several times, adjust the pH of the tris-(2-amino)ethylamine solution to about 11 with sodium hydroxide solution, add the membrane reacted in the previous step, 80°C Reaction 12h;

[0038] (2) Next, methyl acrylate and ethylenediamine are gradua...

Embodiment 2

[0045] Example 2: Hyperbranched Immobilized Metal Affinity Membrane For Cu 2+ adsorption

[0046] Use pH=5 NaAc-HAc buffer to prepare copper ion solutions with different concentrations from low to high (0.1, 0.2, 0.3, 0.5, 1, 2, 3, 5, 10 mM / L), and place a piece of synthesized membrane on In a 50 mL centrifuge tube, add 5 mL of copper ion solution and absorb in a shaker at room temperature for 12 h. 2+ The membrane was repeatedly washed with pH=5 NaAc-HAc buffer and water to remove physically adsorbed Cu on the membrane surface 2+ , the treated membrane was eluted with 10 mL 0.1M EDTA until the surface color of the membrane changed from blue to colorless, and the Cu in the eluate was measured by AAS 2+ concentration. The synthesized hyperbranched immobilized metal affinity membrane for Cu 2+ The adsorption capacity can reach 3.4 μmol / cm 2 .

[0047] like figure 2 As shown, the adsorption of Cu 2+ The original film before and after, the non-hyperbranched control film, ...

Embodiment 3

[0048] Example 3: Hyperbranched Immobilized Metal Affinity Membrane for Protein Adsorption

[0049] Using lysozyme as a model protein, the adsorption isotherms of hyperbranched immobilized metal affinity membrane and control membrane were explored. First, put the synthesized membrane in a centrifuge tube, buffer with 20mM PBS+0.2M NaCl pH=7.4 buffer for half an hour, then add 5mL of lysozyme solution of different concentrations (20mM PBS+0.2M NaCl pH=7.4 The buffer solution was prepared, the concentrations were 0.3, 0.5, 1, 2, 3, 4, 5, 6, 7, 8mg / mL), 25°C, 150r / min in a constant temperature shaker for 6h to adsorption equilibrium. The protein concentration in the supernatant after adsorption was measured with a UV-Vis spectrophotometer at 280 nm. like image 3 As shown, the maximum adsorption capacity of the modified membrane for lysozyme is 162 mg / cm 3 , which is higher than the reported value in the literature and the adsorption capacity of non-hyperbranched affinity memb...

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Abstract

The invention discloses a hyperbranched fixed metal affinity chromatography stationary phase represented by the general structural formula (I), which uses a hyperbranching method on the surface of the chromatography stationary phase to modify the material surface, thereby increasing the density of functional groups on the surface of the material and improving the Protein has a higher adsorption capacity. The chromatography stationary phase prepared by the invention has metal chelation chromatography separation performance. When it is used to separate lysozyme in egg white, good separation effect and high protein recovery rate can be achieved. .

Description

technical field [0001] The invention relates to a hyperbranched fixed metal ion affinity chromatography stationary phase and a preparation method thereof. Background technique [0002] As the material basis of life, protein is of great significance to life activities. With the development of biotechnology, the separation and purification of some medical products such as antibiotics, vaccines, antibodies, peptides, therapeutic proteins, etc. has become more and more important. However, biological macromolecules such as proteins generally exist in the form of complex mixtures, and have special properties, such as sensitivity to temperature, pH, organic solvents, etc., and separation based on their physical and chemical properties can hardly achieve good results. Affinity chromatography is a method of modifying the appropriate ligand on the separation medium to specifically bind to the protein to achieve protein purification. It has high specificity and is widely used. As a k...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01J20/286B01J20/30B01D15/38C07K1/22C01B25/37
Inventor 符瑞卫引茂
Owner NORTHWEST UNIV
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